Myotonia levior is a chloride channel disorder

The group of dominant non-dystrophic myotonias, comprising disorderscharacterized by clinically similar forms of myogenic musclestiffness, is genetically inhomogeneous. Dominant myotonia congenita(Thomsen's disease) is linked to CLCN1, the gene encoding themajor muscle chloride channel, localized on chromosome 7q35.In contrast, dominant myotonias sensitive to potassium are causedby point mutations in SCN4A on chromosome 17q, the gene forthe     

Proof of a non-functional muscle chloride channel in recessive myotonia congenita (Becker) by detection of a 4 base pair deletion

Recessive myotonia congenlta (Becker) is genetically linkedto HUMCLC, the gene encoding the muscular chloride channel,localized on chromosome 7q35. Three point mutations have sofar been reported In HUMCLC, one causing recessive Becker-typemyotonia, the others causing the clinically similar Thomsen-typemyotonia, which is Inherited as a dominant trait. We reporta homozygous patient having a 4 base pair deletion in HUMCLCthat shifts the reading frame and causes early stop codons,thus destroying the gene's coding potential for several membrane-spanningdomains. In addition, we report a patient homozygous for a novelpoint mutation located at the extracellular side of the firstmembrane-spanning domain that causes removal of a negative charge(aspartic acid-136-glyclne). Both mutations lead to the recessivetype of myotonia congenita. Since the patient having the deletionpresents less severe clinical myotonia than the patient carryingthe missense mutation, it seems that the absence or truncationof the channel protein may disturb muscle fibre function lessthan the substitution of a single amino acid.     

Conformation-dependent regulation of inward rectifier chloride channel gating by extracellular protons

We have investigated the gating properties of the inward rectifier chloride channel (Cl_ir) from mouse parotid acinar cells by external protons (H⁺_o) using the whole-cell patch-clamp technique. Increasing the pH_o from 7.4 to 8.0 decreased the magnitude of Cl_ir current by shifting the open probability to more negative membrane potentials with little modification of the activation kinetics. The action of elevated pH was independent of the conformational state of the channel. The effects of low pH on Cl_ir channels were dependent upon the conformational state of the channel. That is, application of pH 5.5 to closed channels essentially prevented channel opening. In contrast, application of pH 5.5 to open channels actually increased the current. These results are consistent with the existence of two independent protonatable sites: (1) a site with a pK near 7.3, the titration of which shifts the voltage dependence of channel gating; and (2) a site with pK = 6.0. External H⁺ binds to this latter site (with a stoichiometry of two) only when the channels are closed and prevent channel opening. Finally, block of channels by Zn²⁺ and Cd²⁺ was inhibited by low pH media. We propose that mouse parotid Cl_ir current has a bimodal dependence on the extracellular proton concentration with maximum activity near pH 6.5: high pH decreases channel current by shifting the open probability to more negative membrane potentials and low pH also decreases the current but through a proton-dependent stabilization of the channel closed state.     

Skeletal muscle sodium channel gating in mice deficient in myotonic dystrophy protein kinase

Myotonic dystrophy, a progressive autosomal dominant disorder, is associated with an expansion of a CTG repeat tract located in the 3'-untranslated region of a serine/threonine protein kinase, DMPK. DMPK modulates skeletal muscle Na channels in vitro, and thus we hypothesized that mice deficient in DMPK would have altered muscle Na channel gating. We measured macroscopic and single channel Na currents from cell-attached patches of skeletal myocytes from mice heterozygous (DMPK(+/-)) and homozygous (DMPK(-/-)) for DMPK loss. In DMPK(-/-) myocytes, Na current amplitude was reduced because of reduced channel number. Single channel recordings revealed Na channel reopenings, similar to the gating abnormality of human myotonic muscular dystrophy (DM), which resulted in a plateau of Na current. The gating abnormality deteriorated with increasing age. In DMPK(+/-) muscle there was reduced Na current amplitude and increased Na channel reopenings identical to those in DMPK(-/-) muscle. Thus, these mouse models of complete and partial DMPK deficiency reproduce the Na channel abnormality of the human disease, providing direct evidence that DMPK deficiency underlies the Na channel abnormality in DM.     

Effect of an N-terminus deletion on voltage-dependent gating of the ClC-2 chloride channel

ClC-2, a chloride channel widely expressed in mammalian tissues, is activated by hyperpolarisation and extracellular acidification. Deletion of amino acids 16-61 in rat ClC-2 abolishes voltage and pH dependence in two-electrode voltage-clamp experiments in amphibian oocytes. These results have been interpreted in terms of a ball-and-chain type of mechanism in which the N-terminus would behave as a ball that is removed from an inactivating site upon hyperpolarisation. We now report whole-cell patch-clamp measurements in mammalian cells showing hyperpolarization-activation of rClC-2Δ16-61 differing only in presenting faster opening and closing kinetics than rClC-2. The lack of time and voltage dependence observed previously was reproduced, however, in nystatin-perforated patch experiments. The behaviour of wild-type rClC-2 did not differ between conventional and nystatin-perforated patches. Similar results were obtained with ClC-2 from guinea-pig. One possible explanation of the results is that some diffusible component is able to lock the channel in an open state but does so only to the mutated channel. Alternative explanations involving the osmotic state of the cell and cytoskeleton structure are also considered. Low extracellular pH activates the wild-type channel but not rClC-2Δ16-61 when expressed in oocytes, a result that had been interpreted to suggest that protons affect the ball-and-chain mechanism. In our experiments no difference was seen in the effect of extracellular pH upon rClC-2 and rClC-2Δ16-61 in either recording configuration, suggesting that protons act independently from possible effects of the N-terminus on gating. Our observations of voltage-dependent gating of the N-terminal deleted ClC-2 are an argument against a ball-and-chain mechanism for this channel.     

Aging and chloride channel regulation in rat fast-twitch muscle fibres

By the use of pharmacological tools, we tested the hypothesis that age-related alterations in the regulatory pathways of chloride channels might contribute to the lowered chloride conductance (G _Cl) found in skeletal muscle of aged rats. The restingG _Cl of extensor digitorum longus (EDL) muscles from adult rats either young (3–4 months old) or aged (29 months old) was measured by means of computerized intracellular microelectrode recordings. In EDL muscle from 3 to 4-month-old rats, 4--phorbol 12,13-dibutyrate (4--PDB), a direct activator of protein kinase C (PKC), decreasedG _Cl in a concentration-dependent manner. The same effect was exerted by cholera toxin. The effects of both the phorbol ester and cholera toxin were inhibited by staurosporine, thus indicating that either direct or indirect (via G protein) activation of PKC accounts for the decrease ofG _Cl. An increase of cytosolic Ca²⁺ by the ionophore A23187 also significantly decreasedG _Cl by 25%. In EDL muscles from aged rats, 4--PDB was 20-fold more potent in blockingG _Cl than in muscles from younger controls, and the ionophore blockedG _Cl by 40%. On the other hand, cholera toxin was ineffective. Our findings support the hypothesis that in fast-twitch muscle the regulation of chloride channels by PKC and Ca²⁺ is a target of the aging process.     

Calcium dependent gating of thel-glutamate activated,excitatory synaptic channel on crayfish muscle

Excitatory, glutamate-activated single channel currents were measured in outside-out patches of crayfish muscle. The open time of single channel openings, and the durations and rates of bursts were evaluated. These kinetic parameters were not appreciably affected by replacement of extracellular Na⁺ by Li⁺ or choline. Changes in extracellular Ca²⁺ concentration Ca_o also did not influence the duration of single openings. However the mean burst duration decreased for Ca_o<13.5 mM and the rate of bursts declined with a power of almost 2 in low Ca_o. At Ca_o<1 mM practically no channel openings were observed in presence of glutamate. In order to exclude more rapid desensitization of the glutamate receptors in low Ca_o as the cause of disappearance of channel openings, glutamate was applied in short pulses with a liquid-filament switch. In 0 Ca_o also a glutamate pulse did not trigger channel openings. In presence of 13.5 mM Ca_o, the inorganic Ca-channel blockers La³⁺ and Cd²⁺ diminished the duration and rate of bursts of channel openings in a similar manner as low Ca_o. The effects of low Ca_o and of Cd²⁺ were tested also on quantal postsynaptic currents, EPSCs, which were recorded through a perfused macro-patch-clamp electrode. At 1.4 mM Ca_o in the perfused electrode tip, spontaneous EPSCs were reduced at least by a factor of 4, and elicited EPSCs by a factor of 16. Application of Cd²⁺ had similarly strong effects on the EPSCs. Also the decay of EPSCs was shortened substantially in 1.4 mM Ca_o or 5 mM Cd²⁺.The inhibitory Cl^–-channel of crayfish muscle, activated by glutamate or GABA, also was studied in outside-out patches. The openings of this channel persisted in 0 Ca_o solutions; the block of channel openings in low Ca_o thus is a specific property of the excitatory channel. The action of Ca_o on the excitatory channel may be described as that of a cofactor to glutamate. A possible reaction scheme is proposed.Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 220     

Severe neonatal non-dystrophic myotonia secondary to a novel mutation of the voltage-gated sodium channel (SCN4A) gene

We report on a patient with a severe, rare neonatal form of non-dystrophic myotonia. The patient presented with facial dysmorphism, muscle hypertrophy, severe constipation, psychomotor delay, and frequent cold-induced episodes of myotonia and muscle weakness leading to severe hypoxia and loss of consciousness. Muscle biopsy was non-specific and electromyography revealed intense generalized myotonia. The myotonic episodes improved after introducing oral mexiletine and maintaining room temperature at 28 degrees C. The patient died at 20 months of age following a bronchopulmonary infection. A previously undescribed de novo heterozygous c.3891C > A change, which predicts p.N1297K in the SCN4A gene. Mutations within the voltage-gated sodium channel alpha-subunit gene (SCN4A) have been described in association with several phenotypes including paramyotonia congenita, hyperkalemic or hypokalemic periodic paralysis, and potassium-aggravated myotonias. The cold-sensitive episodes of stiffness followed by weakness suggested the diagnosis of channelopathy in our patient. However, her neonatal onset, the triggering of severe episodes by exposure to modest decreases in temperature, involvement of respiratory muscles with prolonged apnea, early-onset muscle hypertrophy, psychomotor retardation, and fatal outcome are evocative of a distinct clinical subtype. Our observation expands the phenotypic spectrum of sodium channelopathies.     

A patch-clamp study on a novel γ-aminobutyric acid activated chloride channel of crayfish deep extensor abdominal muscle

The patch-clamp technique was used to study the effect of rapid pulses of γ-aminobutyric acid (GABA) on excised membrane-patches in the outside-out configuration of the deep extensor abdominal muscle (DEAM) of crayfish. Channel currents reversed at the equilibrium potential of Cl⁻ and were blocked by picrotoxin. Rare channel openings were elicited by 0.1 mM GABA, and a saturating open probability of 0.9 was reached with 10 mM GABA. The investigated channel was only sensitive to GABA and is different from a previously described GABAergic channel in crayfish that is in addition sensitive to acetylcholine and glutamate and shows three subconductance states.     

Identification of five new mutations and three novel polymorphisms in the muscle chloride channel gene (CLCN1) in 20 Italian patients with dominant and recessive myotonia congenita. Mutations in brief no. 118. Online

Autosomal dominant myotonia congenita or Thomsen's disease and autosomal recessive myotonia congenita or Becker's are rare nondystrophic disorders due to allelic mutations of the muscle chloride channel gene, CLCN1. We have analysed all 24 exons of the CLCN1 gene, in a panel of 20 unrelated patients (9 with dominant and 11 with recessive mytotonia congenita). We have found five novel mutations including two missense (V5631, F708L), one nonsense (C481X), one splicing (IVS19+2T->A), and one frameshift (2264delC), and also detected the recurrent R894X mutation. These account for 10 of the 22 recessive alleles examined, while no mutations were found in the dominant form. We report three novel polymorphisms (-134T/G, 898C/A and 2154T/C). Our results support high molecular heterogeneity of these myotonias in Italian population and provide new insight for the diagnosis and genetic counselling of these diseases.     

Ca2+-mediated suppression of the GABA-response through modulation of chloride channel gating in frog sensory neurones

A suppressant effect of intracellular free Ca2+ on the gamma-aminobutyric acid (GABA)-induced chloride inward current (ICI(GABA)) was studied in isolated frog sensory neurones under whole cell voltage clamp. Voltage-dependent Ca2+ influx elicited during the steady state of ICI(GABA)-induced a fast, slowly recovering current relaxation in the outward direction, the amplitude of which was dependent on total Ca2+ influx. This suppressant effect showed specificity for different divalent cations, suggesting action at a specific intracellular effector. Single channel recording revealed a Ca2+-dependent decrease in the duration of the open time of the GABA-gated Cl- channel without change in single channel conductance.     

Myotonia caused by mutations in the muscle chloride channel gene CLCN1

Pure non-syndromic, non-dystrophic myotonia in humans is caused by mutations in the genes coding for the skeletal muscle sodium channel (SCN5A) or the skeletal muscle chloride channel (CLCN1) with similar phenotypes. Chloride-channel myotonia can be dominant (Thomsen-type myotonia) or recessive (Becker-type myotonia). More than 60 myotonia-causing mutations in the CLCN1 gene have been identified, with only a few of them being dominant. A common phenotype of dominant mutations is a dominant negative effect of mutant subunits in mutant-WT heterodimers, causing a large shift of the steady-state open probability voltage-dependence towards more positive, unphysiological voltages. The study of the properties of disease causing mutations has helped in understanding the functional properties of the CLC-1 channel that is part of a nine-member gene family of chloride channels. The large body of knowledge obtained for CLC-1 may also help to better understand the other CLC channels, three of which are also involved in genetic diseases.     

Ryanodine modification of RyR1 retrogradely affects L-type Ca2+ channel gating in skeletal muscle

In skeletal muscle, there is bidirectional signalling between the L-type Ca²⁺ channel (1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine-sensitive Ca²⁺ release channel (RyR1) of the sarcoplasmic reticulum (SR). In the case of “orthograde signalling” (i.e., excitation-contraction coupling), the conformation of RyR1 is controlled by depolarization-induced conformational changes of the DHPR resulting in Ca²⁺ release from the SR. “Retrograde coupling” is manifested as enhanced L-type current. The nature of this retrograde signal, and its dependence on RyR1 conformation, are poorly understood. Here, we have examined L-type currents in normal myotubes after an exposure to ryanodine (200 μM, 1 h at 37°C) sufficient to lock RyR1 in a non-conducting, inactivated, conformational state. This treatment caused an increase in L-type current at less depolarized test potentials in comparison to myotubes similarly exposed to vehicle as a result of a ~5 mV hyperpolarizing shift in the voltage-dependence of activation. Charge movements of ryanodine-treated myotubes were also shifted to more hyperpolarizing potentials (~13 mV) relative to vehicle-treated myotubes. Enhancement of the L-type current by ryanodine was absent in dyspedic (RyR1 null) myotubes, indicating that ryanodine does not act directly on the DHPR. Our findings indicate that in retrograde signaling, the functional state of RyR1 influences conformational changes of the DHPR involved in activation of L-type current. This raises the possibility that physiological regulators of the conformational state of RyR1 (e.g., Ca²⁺, CaM, CaMK, redox potential) may also affect DHPR gating.